In recent years, it has become clear that many organisms exploit the base-pairing potential of RNA and DNA to enable sequence-based resistance mechanisms against viruses and other mobile genetic elements. The best known of these mechanisms, RNA interference, uses double-stranded RNA to trigger the silencing of specific genes. However, this mechanism has thus far only been documented in eukaryotes. More recently, clustered regularly interspaced short palindromic repeat (CRISPR) loci, present in the genomes of many eubacteria and nearly all archaebacteria, have been shown to confer sequence-based immunity against bacteriophages. CRISPR loci are accompanied by a set of cas (CRISPR-associated) genes that are likely to encode protein components of the underlying enzymatic machinery. However, the biochemical mechanism of CRISPR- and cas-directed interference is unknown. We propose to dissect the molecular basis for CRISPR and cas gene function. Genome database searches have revealed the presence of a relatively simple CRISPR/cas locus in a strain of Staphylococcus epidermidis, and the sequence of the locus suggests that it specifies resistance not only to bacteriophages but also to staphylococcal conjugative plasmids. Given the clinical importance of staphylococci and the experimental tractability of S. epidermidis, we will use it as a model system to explore fundamental aspects of CRISPR-derived immunity in eubacteria. Preliminary results confirm that an S. epidermidis strain carrying the CRISPR locus is defective as a plasmid conjugation recipient, whereas an isogenic strain lacking the CRISPR locus is not. These and other observations confirm a role for CRISPR loci in restricting horizontal gene transfer in eubacteria, and provide us with a simple and convenient assay for CRISPR function. We will use this system to conduct a genetic analysis of CRISPR and cas gene function. In particular, we will define the sequence characteristics of both the CRISPR locus and the target plasmid that are needed for interference, and we will test the involvement of specific cas genes in this process. In addition, we will conduct preliminary biochemical analyses of the previously reported CRISPR transcripts. The results of these experiments will place critical constraints on viable models of CRISPR/cas function, and will set the stage for in-depth mechanistic analyses. S. epidermidis and Staphylococcus aureus are the most common causes of nosocomial infections, and the transfer of plasmids that carry antimicrobial resistance genes contributes to the ever-worsening spread of these pathogens. Understanding CRISPR function is an important step in the development of therapeutic interventions that exploit this pathway to impede the spread of antibiotic resistance. In addition, given the important role of bacteriophages in the evolution of pathogenic bacteria, the study of CRISPR function will improve our understanding of how infectious diseases emerge, disappear and re-emerge. PUBLIC HEALTH RELEVANCE: Clustered regularly interspaced short palindromic repeat (CRISPR) loci confer acquired, sequence-based resistance against viruses and conjugative plasmids in many eubacteria and nearly all archaebacteria, but the underlying mechanisms are unknown. The transfer of antibiotic resistance genes on conjugative plasmids contributes to the spread of pathogenic bacterial strains, leading to significant threats to human health. The proposed studies will clarify the mechanisms of CRISPR function, and will therefore contribute to our ability to exploit this natural pathway to prevent and treat infectious disease.